Anti-viral defense by an ADP-ribosyltransferase that targets mRNA to block translation (RIP-Seq in vitro)

Host-pathogen conflicts are crucibles of molecular innovation. Selection for immunity to pathogens has driven the evolution of sophisticated immunity mechanisms throughout biology, including in bacteria that must evade their viral predators known as bacteriophages. Here, we characterize a toxin-antitoxin-chaperone system, CmdTAC, in Escherichia coli that provides robust defense against infection by T4 phage. During infection, newly synthesized capsid protein triggers dissociation of the chaperone CmdC from the CmdTAC complex, leading to destabilization and degradation of the antitoxin CmdA, with consequent liberation of the toxin CmdT, an ADP-ribosyltransferase. Strikingly, CmdT does not target a protein, DNA, or structured RNA, the known targets of other ADP-ribosyltransferases. Instead, CmdT modifies the N6 position of adenine in GA dinucleotides within single-stranded RNAs to robustly block mRNA translation and viral replication. Our work reveals both a new mechanism of anti-phage defense and a new class of ADP-ribosyltransferases that targets mRNA. The goal of this experiment is to identify transcripts modified by the ADP-ribosyltransferase CmdT with an in vitro approach. CmdT was incubated with RNA extracted from E. coli infected with the bacteriophage T4 and supplied biotinylated NAD+ and then a streptavidin pulldown was performed on the RNA to identify modified transcripts.

宿主与病原体的冲突是分子创新的催生熔炉。针对病原体免疫的选择压力，推动了整个生物界复杂免疫机制的演化，其中也包括必须躲避自身病毒捕食者——噬菌体（bacteriophage）的细菌。本研究对大肠杆菌（Escherichia coli）中的CmdTAC毒素-抗毒素-分子伴侣系统（toxin-antitoxin-chaperone system）进行了表征，该系统可对T4噬菌体（T4 phage）感染提供强效防御。在感染过程中，新合成的衣壳蛋白会触发分子伴侣CmdC从CmdTAC复合物中解离，进而导致抗毒素CmdA发生不稳定并被降解，最终释放出作为ADP-核糖基转移酶（ADP-ribosyltransferase）的毒素CmdT。值得注意的是，CmdT并不靶向其他ADP-核糖基转移酶已知的蛋白质、DNA或结构化RNA底物。与之相反，CmdT会对单链RNA中GA二核苷酸内腺嘌呤的N6位进行修饰，从而强效阻断mRNA翻译与病毒复制。本研究不仅揭示了一种全新的抗噬菌体防御机制，还发现了一类靶向mRNA的新型ADP-核糖基转移酶。本实验的研究目标是通过体外实验方法，鉴定被ADP-核糖基转移酶CmdT修饰的转录本。具体实验方案为：将CmdT与从感染T4噬菌体的大肠杆菌中提取的RNA共同孵育，并加入生物素化烟酰胺腺嘌呤二核苷酸（biotinylated NAD+），随后对RNA进行链霉亲和素下拉实验以鉴定被修饰的转录本。